CN110278872B - Walnut somatic embryo rooting and seedling exercising transplanting method - Google Patents

Walnut somatic embryo rooting and seedling exercising transplanting method Download PDF

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CN110278872B
CN110278872B CN201910628721.XA CN201910628721A CN110278872B CN 110278872 B CN110278872 B CN 110278872B CN 201910628721 A CN201910628721 A CN 201910628721A CN 110278872 B CN110278872 B CN 110278872B
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rooting
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CN110278872A (en
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杨克强
段琪程
常董董
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Shandong Agricultural University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
    • A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
    • A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
    • A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
    • A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G31/00—Soilless cultivation, e.g. hydroponics
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants

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Abstract

The invention discloses a walnut somatic embryo rooting and seedling hardening transplanting method, which comprises the following steps: (1) the method comprises the following steps of (1) growth culture of somatic embryos, (2) germination of the somatic embryos, (3) rooting of the somatic embryos, and (4) transplantation of somatic embryo seedlings. The walnut somatic embryo rooting and seedling exercising transplanting method provided by the invention solves the problems of difficult walnut somatic embryo rooting and low transplanting survival rate, and is counted by experiments: the germination rate of the somatic embryos can reach 45.22%, the rooting rate of the germinated somatic embryos can reach 43.43% compared with the prior art, and the transplanting survival rate of the somatic embryos transplanted by using well-grown somatic embryos can reach 57.69%. The method can provide a large amount of seedlings in a short time and can completely meet the requirement of large-scale production.

Description

Walnut somatic embryo rooting and seedling exercising transplanting method
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for rooting and hardening seedling and transplanting a walnut somatic embryo.
Background
Juglans (Juglans) is a tree of Juglandaceae (Jugladaceae) deciduous leaves, and has more than 20 species worldwide, wide geographical distribution, and distribution in Asia, America, and Europe (Forde and McGranahan, 1992). Walnut (Juglans regia L.) is the most economically valuable species of the genus Juglans, and has been cultivated around the world (Forde and McGranahan, 1992). The walnut is originated in China and also is the world's leading country, and its cultivation area is the first world and the yield is the second world (ximeningwu, 1996).
Walnuts have a long cultivation history in China, are high in economic benefit and rich in nutrition, and are also important materials for medical care (Xiyuke, 1996). According to records in compendium of materia medica, walnuts have unique effects of moistening lung, enriching blood, invigorating stomach and dissolving phlegm. Is highly advocated by medical scientists at home and abroad and is a top-quality product (Chen and Li Lei Ke, 2005) for health preserving, health care and life prolonging. Walnut kernel has special effect on brain development, linoleic acid and linolenic acid have higher content in walnut kernel, can purify blood, decompose impurities in blood, improve physiological function of brain (Koufuyan, 2000), is also an ideal cosmetic for human body, and walnut kernel acetone extract also has anti-aging effect (chlorpheniramine and Yi Zheng, 2006). In recent researches, the green dragon skin of the walnut has certain efficacy on prevention and treatment of tumors, and meanwhile, the green dragon skin is also a traditional Chinese medicine (Zhongarmy plum and Liuyumei, 2014) for pain relief and bacteriostasis in the traditional Chinese medicine. Today, the economy is rapidly developing, water pollution and air pollution are extremely serious, and the preparation of activated carbon by walnut shells can effectively prevent and treat the activated carbon (cinnabar, 2015). The walnut nut inner membrane is also a traditional Chinese medicinal material and has the effects of reinforcing the kidney and arresting seminal emission (Zhangxu, 2015). The walnut wood is heavy and hard, resistant to collision, friction, decay and drying, and easy to dry and less in deformation; easy construction and gluing. The wood is hard, suitable for carving parts, can resist various climates, can be applied by any coating method, and is most suitable for manufacturing furniture and interior decoration.
Regarding the germination and seedling formation of somatic embryos of walnuts, the germination efficiency of somatic embryos is relatively low in walnuts (Lee, 1988; Deng and Cornu 1992; Vahdati et al, 2006). Generally, it is difficult to form normal plants, although some somatic embryos can germinate without refrigeration, because the desired germination of somatic embryos requires breaking the dormancy of the shoot tip. Deng et al (1998) can increase the germination rate of somatic embryos by 3-5 times by adopting a dehydration treatment mode. Increasing the concentration of ABA can also improve the germination rate of somatic embryos, and the concentration of ABA is increased to 5.0 mg.L-1The germination rate of somatic embryos can be improved by 6% (Kourosh et al, 2008). Kourosh et al (2008) found that sucrose had little effect on somatic embryo germination by debranning normal and abnormal somatic embryos. The degree of health of somatic embryos also has a significant impact on somatic embryo germination, and somatic embryos that are less healthy are difficult to germinate even after refrigeration and dehydration (Kourosh et al, 2008). Since the somatic embryo has two polarities, and the upper and lower hypocotyls germinate simultaneously, the root does not need to be induced independently, and only a proper culture medium needs to be selected for rooting culture, so that the somatic embryo develops into a complete somatic embryo seedling (Tanghao Ru, etc., 2000). Domestic and foreign researches show that although somatic embryos can germinate, the multiplication coefficient is low, the large-scale industrialized seedling culture and the large-scale market demand cannot be met, the problem that the somatic embryos are difficult to root is not solved, and the method cannot be applied to production at all. Therefore, an efficient and stable walnut somatic embryo germination and rooting system is established, and the problem of walnut germination and rooting is solvedThe problem of difficulty is the difficulty in tissue culture of walnuts at home and abroad at present.
A certain report has been made about induction and rapid propagation of walnut somatic embryos at home and abroad (Tulecke et al, 1985; Kornova et al, 1989; Yuanqiao et al, 1990; Tanghao et al, 2000; Zhangxiang et al, 2011), but the research on hardening, transplanting and domestication of walnut somatic embryo seedlings is less, which is very unfavorable for putting the somatic embryo seedlings into production.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a walnut somatic embryo rooting and seedling hardening transplanting method to solve the problem of difficult seedling formation of walnut somatic embryos.
In order to achieve the purpose, the invention adopts the following technical scheme:
a walnut somatic embryo rooting and seedling exercising transplanting method comprises the following steps:
(1) growth culture of somatic embryos: inoculating walnut secondary somatic embryos into a basic culture medium, culturing at 20-24 ℃ in the dark, and germinating when the somatic embryos grow to 3-4 mm;
(2) somatic embryo germination: inoculating the somatic embryos cultured in the step (1) to a germination culture medium, culturing for 2-3 weeks, then pretreating at a low temperature for 2-3 weeks, then transferring the somatic embryos pretreated at the low temperature to a fresh germination culture medium, performing day and night illumination periodic culture, and germinating after 6-9 days;
(3) rooting of somatic embryos: inoculating the somatic embryo with the upper and lower embryonic axis differentiation after germination into a rooting culture medium, and irradiating for 16 h.d at the illumination intensity of 3000-3500 lx-1Culturing for 12-15 days at 24-26 ℃, and inducing to root;
(4) and (3) transplanting somatic cell culture seedlings: and (4) hardening the somatic embryo seedlings which are cultured in the step (3) and have 3-4 true leaves and good root system development, and transplanting the hardened seedlings into a mixed matrix for culturing.
Preferably, in the step (1), the walnut secondary somatic embryo is obtained by multiplication culture of a walnut primary somatic embryo; proliferation ofThe culture medium used for culturing is supplemented with 1.0 mg. L-1 6-BA、2.0mg·L-1KT、0.01mg·L-1IBA、30g·L-1Sucrose and 7 g.L-1DKW medium of agar.
The length of the walnut secondary somatic embryo is 2 mm.
Preferably, in the step (1), the basic culture medium is added with 20-30 g.L-1Sucrose and 7-8 g.L-1DKW medium of agar, pH 5.6.
Preferably, in step (1), 1 fresh basal medium is inoculated every 1 week during the culture.
Preferably, in the step (2), the germination medium is added with 1.0-3.0 mg-L-1GA3、20～30g·L-1Sucrose and 7-8 g.L-1DKW medium of agar, pH 5.6.
Preferably, in step (2), the conditions of the circadian cycle culture are: light treatment for 16 h.d-1The illumination intensity is 3000-3500 lx, and the temperature is 24-26 ℃.
Preferably, in the step (3), 1.0-3.0 mg.L of the rooting medium is added-1 6-BA、20～30g·L-1Sucrose and 7-8 g.L-1DKW medium of agar, pH 5.6.
Preferably, in step (3), 2-3 individual cell embryos are inoculated into each culture flask containing rooting medium.
Preferably, in the step (4), the hardening-seedling treatment specifically comprises: moving the culture bottle of the somatic embryo seedlings together with the somatic embryo seedlings out of the culture frame, and gradually opening a sealing film of the culture bottle after adapting to 1 d; and taking out the somatic embryo seedlings after 2-3 d, washing the culture medium on the somatic embryo seedlings, and transplanting the somatic embryo seedlings to a position where perlite, plant ash and vermiculite are mixed according to a volume ratio of 1:2:1, thoroughly watering with clear water after transplanting, placing the somatic embryo seedlings in a sunshade net for sunshade, keeping the relative humidity at more than 75%, and hardening the seedlings for 1 week.
Preferably, in the step (4), the ingredients of the mixing matrix are as follows by volume ratio: perlite: plant ash: 1, vermiculite: 2: 1.
the invention has the beneficial effects that:
the walnut somatic embryo rooting and seedling exercising transplanting method provided by the invention solves the problems of difficult walnut somatic embryo rooting and low transplanting survival rate, and is counted by experiments: the germination rate of the somatic embryos can reach 45.22%, the rooting rate of the germinated somatic embryos can reach 43.43% compared with the prior art, and the transplanting survival rate of the somatic embryos transplanted by using well-grown somatic embryos can reach 57.69%. The method can provide a large amount of seedlings in a short time and can completely meet the requirement of large-scale production.
Drawings
FIG. 1: and (3) growing and culturing somatic embryos.
FIG. 2: somatic embryos germinate.
FIG. 3: and (4) rooting and culturing the somatic embryo.
FIG. 4: hardening seedlings at normal temperature.
FIG. 5: and (5) hardening seedlings in a greenhouse.
FIG. 6: greenhouse acclimation (local picture).
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As introduced in the background technology, the somatic embryos of walnuts have the problems of difficult rooting and low transplanting survival rate, and the large-scale production and planting of walnuts are severely restricted.
Based on the above, the invention aims to provide a walnut somatic embryo rooting and seedling hardening transplanting method, so as to solve the problem of difficult seedling formation of walnut somatic embryos.
In one embodiment of the invention, the given walnut somatic embryo rooting and seedling exercising transplanting method comprises the following steps:
(1) growing and culturing somatic embryos, (2) germinating the somatic embryos, (3) rooting the somatic embryos, and (4) transplanting somatic embryo seedlings; wherein:
the method for growing and culturing the somatic embryos in the step (1) comprises the following steps: selecting secondary somatic embryos which are well developed and have the length of 2mm, inoculating the secondary somatic embryos into a basic culture medium on a superclean bench, culturing at 20-24 ℃ in the dark, transferring 1 time of fresh culture medium every 1 week, and germinating when the somatic embryos are developed to 3-4 mm;
the basic culture medium is added with 20-30 g.L-1Sucrose and 7-8 g.L-1DKW medium of agar, pH 5.6.
The somatic embryo germination method in the step (2) is as follows: and (2) inoculating the somatic embryos cultured in the step (1) to a germination culture medium, culturing for 2-3 weeks, and then pretreating at a low temperature of 4 ℃ for 2-3 weeks. Then transferring the somatic embryo into fresh germination culture medium, making day and night illumination periodic culture, and making illumination treatment for 16 h.d-1Germinating the somatic embryos after 6-9 days at the illumination intensity of 3000-3500 lx and the temperature of 25 +/-1 ℃;
the germination culture medium is added with 1.0-3.0 mg-L-1GA3、20～30g·L-1Sucrose and 7-8 g.L-1DKW medium of agar, pH 5.6.
The somatic embryo rooting method in the step (3) comprises the following steps: inoculating the somatic embryos differentiated by the upper and lower embryonic axes in the step (2) into a rooting culture medium, inoculating 2-3 individual cell embryos into each culture bottle, and irradiating for 16 h.d at the illumination intensity of 3000-3500 lx for 16 h.d-1Culturing for 12-15 days at 24-26 ℃, and inducing to root;
the rooting medium is added with 1.0-3.0 mg.L-1 6-BA、20～30g·L-1Sucrose and 7-8 g.L-1DKW medium of agar, pH 5.6.
The method for transplanting the somatic embryo seedlings in the step (4) comprises the following steps: culturing a culture bottle with 3-4 true leaves and good root system development (the good root system development means that the length of the root system is more than 5cm, and the number of branches of the root system is more than 2) somatic embryo seedlings in the step (3), and moving the culture bottle and the somatic embryo seedlings out of the culture frame. After adapting to 1d, the sealing membrane of the culture flask is gradually opened. And 2-3 d later, clamping the somatic embryo seedlings by using tweezers, washing the culture medium on the somatic embryo seedlings by using clear water, wherein the volume ratio of perlite, plant ash and vermiculite is 1:2: the transplanting substrate of 1 is transplanted, clear water is used for thorough watering after the transplanting, the somatic embryo seedlings are placed into a sunshade net for sunshade, good humidity conditions are kept (the relative humidity is kept to be more than 75%), after hardening seedlings for 1 week, the somatic embryo seedlings are transplanted into a mixed substrate for cultivation, and the transplanting survival rate is counted after 3 weeks.
The mixed matrix comprises the following components in volume ratio: perlite: plant ash: 1, vermiculite: 2: 1.
an important feature of somatic embryos is the ability to undergo secondary embryogenesis repeatedly. This property of somatic embryos is exploited not only for rapid propagation but also for genetic transformation. The generation of secondary somatic embryos is restricted by various factors, wherein a plant growth regulator, a nitrogen source, a carbon source and the like are related to the proliferation rate of the somatic embryos and the quality of the secondary embryos.
Germination of a somatic embryo refers to elongation of the hypocotyl and growth and development of roots and/or branches thereof. Generally, somatic embryos are ontogenetically similar to zygotic embryos and develop to maturity through typical globular, torpedo-shaped and cotyledonary stages. Because somatic embryos are not synchronously generated, the developmental maturity of the somatic embryos is not synchronous; moreover, even with respect to synchronously developing somatic embryos, the embryonic organs will mature at different rates. Thus, somatic embryo germination often forms only shoots or roots and not plants. Although many plants can undergo somatic embryogenesis, there are limited plants that are truly ideal somatic embryogenesis systems for propagating plantlets.
The seedling formation of the somatic embryo means that the germinated somatic embryo is transformed into a normally growing plant. The ability to obtain germinated somatic embryos also does not mean that somatic embryo seedlings can be obtained. The walnut somatic embryos germinate only roots, only branches or both roots and branches, and the seedling forming capacities of the roots, the branches and the branches are greatly different.
The germination and seedling rate of the walnut somatic embryos is extremely low, so that the success report of seedling breeding by using the somatic embryogenesis system of the walnut is very little.
The method takes secondary somatic embryos as a culture object, and has the advantages that the number of the secondary embryos is more through multiplication culture compared with that of primary somatic embryos, and the proportion of malformed embryos of the secondary embryos compared with the primary embryos is less. The secondary somatic embryo of the invention is obtained by multiplication culture of the primary somatic embryo, and the composition of the culture medium has great influence on the formation of the walnut secondary somatic embryo. If the composition of the culture medium is not proper, the generation frequency of the secondary embryos is low, and most of the generated secondary embryos are blocked at the globular stage. The composition of the culture medium is optimized, the culture medium is adopted to carry out multiplication culture on the primary somatic embryos, the multiplication rate of the somatic embryos is high, and the generated secondary somatic embryos are tidy in development and strong in growth potential.
In order to improve the germination rate, rooting rate and transplanting survival rate of the somatic embryos of the walnuts, the germination culture medium, the rooting culture medium and the hardening-seedling transplanting method are optimized. The germination culture medium and the rooting culture medium optimized by the method can obviously improve the germination rate and the rooting rate of the walnut somatic embryos, and particularly, the 6-BA with specific concentration is added into the rooting culture medium, so that the walnut secondary somatic embryos can successfully root.
When the tissue-cultured somatic embryo seedlings are transplanted, the water loss rate is high, the seedlings die easily, the seedling hardening time needs to be accurately controlled, and the matrix proportion needs to be accurately mastered. Turfy soil: when the vermiculite is 1:2:1, the transplanting survival rate is highest; when the ratio of the plant ash in the matrix is higher, the air permeability of the matrix is easily reduced, and the transplanting survival rate is reduced.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available. The culture medium used in the examples of the present invention is as follows:
the DKW comprises the following specific components: KNO3 1367.00mg·L-1，NH4NO3 1416.00mg·L-1，MgSO4361.49mg·L-1，KH2PO4 265.00mg·L-1，CaCl2·2H20 112.50mg·L-1，MnSO4·H2O 33.50mg·L-1，ZnSO4·6H2O 17.00mg·L-1，H3BO3 6.20mg·L-1，NaMoO4 0.39mg·L-1，CuSO4·5H2O 0.25mg·L-1，CoCl2·6H2O 0.05mg·L-1，Na2-EDTA 45.40mg·L-1，FeSO4·7H20 33.80mg·L-1。
The basic culture medium refers to: DKW culture medium + 20-30 g.L without any growth regulating substance-1Sucrose + 7-8 g.L-1Agar, pH 5.6.
The germination medium refers to: DKW + 1.0-3.0 mg.L-1GA3+20～30g·L-1Sucrose + 7-8 g.L-1Agar, pH 5.6.
The rooting medium is as follows: DKW + 1.0-3.0 mg.L-1 6-BA+20～30g·L-1Sucrose + 7-8 g.L-1Agar, pH 5.6.
The somatic embryo adopted in the embodiment of the invention is a secondary somatic embryo of a walnut variety 'Ruijia'; the walnut secondary somatic embryo is obtained by performing multiplication culture on a walnut primary somatic embryo; the culture medium used for proliferation culture contains 1.0 mg.L-1 6-BA、2.0mg·L-1KT、0.01mg·L-1IBA、30g·L-1Sucrose and 7 g.L-1DKW medium of agar.
Wherein, the walnut primary somatic embryo is formed by taking the stem section of the test-tube plantlet of the walnut variety 'Ruijia' as an explant through induction.
Example 1:
(1) selecting well-developed walnut secondary somatic embryos with the length of 2mm, inoculating the walnut secondary somatic embryos to a basic culture medium on a super-clean workbench, culturing at 22 ℃ in the dark, transferring 1 time of fresh culture medium every 1 week, and germinating when the somatic embryos grow to 3-4 mm.
(2) Inoculating the somatic embryo cultured in the step (1) into DKW +2.0 mg.L-1GA3+30g·L-1Sucrose +8 g.L-1Agar, pH 5.6 germination medium, after 2 weeks of culture, was subjected to a low temperature pre-treatment at 4 ℃ for 3 weeks. Then transferring the somatic embryo into fresh germination culture medium, making day and night illumination periodic culture, and making illumination treatment for 16 h.d-1The illumination intensity is 3200lx, the temperature is 25 +/-1 ℃, and the somatic embryos germinate after 8 days; the statistical germination rate can reach 45.22%.
(3) Inoculating normally germinated somatic embryos to DKW +2.0 mg-L-1 6-BA+20g·L-1Sucrose +8 g.L-1Agar, rooting culture medium with pH of 5.6, sterilizing the culture medium with autoclave at 121 deg.C for 20min, subpackaging in glass culture bottles, inoculating 3 individual cell embryos in each culture bottle, culturing in day and night light at illumination intensity of 3000lx for 16 h.d-1Rooted somatic embryos were obtained after two weeks at 25 ℃. The rooting rate was 43.43%.
(4) Culturing a culture bottle with 3-4 true leaves and good root system development (the good root system development means that the length of the root system is more than 5cm, and the number of branches of the root system is more than 2) somatic embryo seedlings in the step (3), and moving the culture bottle and the somatic embryo seedlings out of the culture frame. After adapting to 1d, the sealing membrane of the culture flask is gradually opened. And 2d, clamping the somatic embryo seedlings by using forceps, washing the culture medium on the somatic embryo seedlings by using clear water, wherein the volume ratio of perlite, plant ash and vermiculite is 1:2: the transplanting substrate of 1 is transplanted, the transplanting substrate is thoroughly watered with clear water after being planted, the somatic embryo seedlings are put into a sunshade net for sunshade, good humidity conditions are kept (the relative humidity is kept above 75 percent), after hardening for a week, the somatic embryo seedlings are transplanted into a mixed substrate for culture, and the mixed substrate comprises the following components in volume ratio: perlite: plant ash: 1, vermiculite: 2: 1; after 3 weeks, the statistical survival rate of the transplantation is 57.69%.
Comparative example 1:
well-developed somatic cells with mature architecture that developed to 4mm were selectedEmbryo, inoculating it into DKW +2.0 mg.L-1GA3+30g·L-1Sucrose +8 g.L-1Agar, germination medium with pH of 5.6, and sterile medium in sterile culture dish at 121 deg.C for 20 min. Sealing the inoculated culture dish with sealing film, inoculating somatic embryo into fresh germination culture medium after germination treatment, and culturing day and night with illumination intensity of 3000lx and illumination time of 16 h.d-1And the germination rate of the somatic embryo can reach 35.69 percent after 9 days at the temperature of 25 ℃.
Inoculating normally germinated somatic embryos to DKW +2.0 mg-L-1 6-BA+20g·L-1Sucrose +8 g.L-1Agar, rooting culture medium with pH of 5.6, sterilizing the culture medium with autoclave at 121 deg.C for 20min, subpackaging in glass culture bottles, inoculating 3 individual cell embryos in each culture bottle, culturing in day and night light at illumination intensity of 3000lx for 16 h.d-1Rooted somatic embryos were obtained after two weeks at 25 ℃. The rooting rate is 40.10%.
The method is characterized by comprising 3-4 true leaves, and well-developed somatic embryo seedlings are prepared by mixing perlite, plant ash and vermiculite in a matrix ratio of 1: 3: 1, transplanting the seedlings, dividing 30 seedlings into 3 groups, watering the seedlings with clear water after planting, putting the somatic embryo seedlings into a sunshade net for sunshade, keeping a good humidity condition, hardening the seedlings for 1 week, transplanting the seedlings into a mixed matrix for culture, and counting the transplanting survival rate to 42.43% after 3 weeks.
Comparative example 2:
2mm somatic embryos are selected and inoculated into DKW culture medium +30 g.L without any growth regulating substances-1Sucrose +8 g.L-1Agar, basic culture medium with pH value of 5.6, sterilizing the culture medium with autoclave at 121 deg.C for 20min, and subpackaging in sterile culture dish. After inoculation, the culture dish was sealed with a sealing film, cultured at 22 ℃ in the dark, and the culture medium was transferred 1 time every 1 week. And (3) carrying out low-temperature germination pretreatment when the somatic embryo grows to 5mm, so as to break the dormant state of the somatic embryo, wherein the germination rate is 23.75%.
Selecting a well-developed 5 mm-developed mature structureThe somatic embryo of (3), which was inoculated into DKW +1.0 mg. L-1GA3+30g·L-1Sucrose +8 g.L-1Agar, germination medium with pH of 5.6, and sterile medium in autoclave at 121 deg.C for 20 min. Sealing the inoculated culture dish with sealing film, inoculating somatic embryo into fresh germination culture medium after germination treatment, and culturing day and night with illumination intensity of 3000lx and illumination time of 16 h.d-1And the somatic embryo germinates at 25 ℃ after 9 days, and the germination rate is 21.04%.
Inoculating normally germinated somatic embryos to DKW +3.0 mg-L-1 6-BA+30g·L-1Sucrose +7 g.L-1Agar, rooting culture medium with pH of 5.6, sterilizing the culture medium with autoclave at 121 deg.C for 20min, subpackaging in glass culture bottles, inoculating 3 individual cell embryos in each culture bottle, culturing in day and night light at illumination intensity of 3000lx for 16 h.d-1Rooted somatic embryos were obtained after two weeks at 25 ℃. The rooting rate can reach 34.59%.
The method is characterized by comprising 3-4 true leaves, and well-developed somatic embryo seedlings are prepared by mixing perlite, plant ash and vermiculite in a matrix ratio of 1:2:1, transplanting the seedlings, dividing 30 seedlings into 3 groups, watering the seedlings with clear water after planting, putting the somatic embryo seedlings into a sunshade net for sunshade, keeping a good humidity condition, hardening the seedlings for 1 week, transplanting the seedlings into a mixed matrix for culture, and counting the transplanting survival rate to 57.69% after 3 weeks.
Comparative example 3:
selecting 1mm somatic embryo to inoculate into DKW culture medium without any growth regulating substance +25 g.L-1Sucrose +7 g.L-1Agar, basic culture medium with pH value of 5.6, sterilizing the culture medium with autoclave at 121 deg.C for 20min, and subpackaging in sterile culture dish. After inoculation, the culture dish was sealed with a sealing film, cultured at 22 ℃ in the dark, and the culture medium was transferred 1 time every 1 week. And (3) germinating and pretreating the somatic embryos at normal temperature when the somatic embryos grow to 4mm, and counting that the germination rate of the somatic embryos which are not subjected to refrigerated germination pretreatment is 20.22%.
Selecting a tool which grows to 4mmWell-developed somatic embryos with mature structures were inoculated to DKW +3.0 mg.L-1GA3+30g·L-1Sucrose +7 g.L-1Agar, a germination culture medium with the pH value of 5.6, sterilizing the culture medium by using an autoclave at 121 ℃ for 20min, subpackaging the sterilized culture medium in a sterile culture dish, inoculating the sterilized culture dish, sealing the culture dish by using a sealing film, inoculating somatic embryos into a fresh germination culture medium after the germination treatment, and carrying out day and night illumination periodic culture, wherein the illumination intensity is 3000lx, and the illumination time is 16 h.d-1And the somatic embryo germinates at 25 ℃ after 9 days, and the germination rate is 36.14%.
Inoculating normally germinated somatic embryos to DKW +1.0 mg-L-1 6-BA+30g·L-1Sucrose +7 g.L-1Agar, rooting culture medium with pH of 5.6, sterilizing the culture medium with autoclave at 121 deg.C for 20min, subpackaging in glass culture bottles, inoculating 2 individual cell embryos in each culture bottle, culturing in day and night light at illumination intensity of 3000lx for 16 h.d-1And obtaining the rooted somatic embryo seedlings after 17 days at the temperature of 25 ℃. The rooting rate can reach 25.63%.
The method is characterized by comprising 3-4 true leaves, and well-developed somatic embryo seedlings are prepared by mixing perlite, plant ash and vermiculite in a matrix ratio of 1: 4: 1, transplanting the seedlings, dividing 30 seedlings into 3 groups, watering the seedlings with clear water after planting, putting the somatic embryo seedlings into a sunshade net for sunshade, keeping a good humidity condition, hardening the seedlings for 1 week, transplanting the seedlings into a mixed matrix for culture, and counting the transplanting survival rate to be 28.87% after 3 weeks.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (5)

1. A walnut somatic embryo rooting and seedling exercising transplanting method is characterized by comprising the following steps:
(1) growth culture of somatic embryos: inoculating walnut secondary somatic embryos into a basic culture medium, culturing at 20-24 ℃ in the dark, and germinating when the somatic embryos grow to 3-4 mm;
(2) somatic embryo germination: inoculating the somatic embryos cultured in the step (1) to a germination culture medium, culturing for 2-3 weeks, then pretreating at a low temperature for 2-3 weeks, then transferring the somatic embryos pretreated at the low temperature to a fresh germination culture medium, performing day and night illumination periodic culture, and germinating after 6-9 days;
(3) rooting of somatic embryos: inoculating the somatic embryo with the upper and lower embryonic axis differentiation after germination into a rooting culture medium, and irradiating for 16 h.d at the illumination intensity of 3000-3500 lx-1Culturing for 12-15 days at 24-26 ℃, and inducing to root;
(4) transplanting somatic embryo seedlings: hardening the somatic embryo seedlings which are cultured in the step (3) and have 3-4 true leaves and good root system development, and transplanting the hardened seedlings into a mixed matrix for culturing;
in the step (1), the walnut secondary somatic embryo is obtained by multiplication culture of a walnut primary somatic embryo; the culture medium used for proliferation culture contains 1.0 mg.L-16-BA、2.0mg·L-1KT、0.01mg·L-1IBA、30g·L-1Sucrose and 7 g.L-1DKW medium of agar;
in the step (1), 20-30 g.L of basic culture medium is added-1Sucrose and 7-8 g.L-1DKW medium of agar, pH 5.6;
in the step (2), 1.0-3.0 mg.L of germination culture medium is added-1GA3、20～30g·L-1Sucrose and 7-8 g.L-1DKW medium of agar, pH 5.6;
in the step (3), 1.0-3.0 mg.L of the rooting culture medium is added-16-BA、20～30g·L-1Sucrose and 7-8 g.L-1DKW medium of agar, pH 5.6;
in the step (4), the seedling exercising treatment specifically comprises the following steps: moving the culture bottle of the somatic embryo seedlings together with the somatic embryo seedlings out of the culture frame, and gradually opening a sealing film of the culture bottle after adapting to 1 d; and 2-3 d, taking out the somatic embryo seedlings, washing the culture medium on the somatic embryo seedlings, transplanting the somatic embryo seedlings into a transplanting matrix consisting of perlite, plant ash and vermiculite according to the volume ratio of 1:2:1, thoroughly watering the transplanted somatic embryo seedlings with clear water, putting the somatic embryo seedlings into a sunshade net for sunshade, keeping the relative humidity above 75%, and hardening the seedlings for 1 week.
2. The method according to claim 1, wherein in step (1), 1 transfer of fresh basal medium is performed every 1 week during the culture.
3. The method according to claim 1, wherein in step (2), the conditions of the circadian cycle culture are: light treatment for 16 h.d-1The illumination intensity is 3000-3500 lx, and the temperature is 24-26 ℃.
4. The method of claim 1, wherein in step (3), 2-3 individual cell embryos are inoculated into each culture flask containing rooting medium.
5. The method according to claim 1, wherein in the step (4), the ingredients of the mixing matrix are as follows by volume ratio: perlite: plant ash: 1, vermiculite: 2: 1.
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